Chromatography Forum

Hello,

I am a confused biology student that is way out of my depth in an organic chemistry lab!

I am performing SPE on dilute cell cultures (1 ml culture diluted in 100 ml milliQ) followed by GCMS to quantify concentrations of a certain metabolite. I have constructed a nice straight forward calibration curve using milliQ spiked with a known concentrations of standard, but now I want to check for possible matrix effects.

The way I understand it is that I could run 1 ml of my media, plus 99 ml milliQ, plus spiking solutions of known concentrations and I should get a gradient identical to the previous calibration curve I constructed, is this right? However, the cells I am growing in the media will also be producing other metabolites along side the ones I'm interested in, surely these could also contribute towards matrix effects? Is there any way I can check for this? Would it work if I ran 1 ml of culture, plus 99 ml milliQ, plus a range of spiking solutions and see if the gradient of the line is the same as in the original calibration curve?

Any help would be much appreciated, thanks.

You're on the right track, but you're conflating two different issues: matrix effects and method specificity.

What you are doing is referred to as "standard additions" analysis. You run the calibration plot with standards only and with standards spiked into your matrix. You should get the same slope in both cases, but the Y-intercept of the spike plot should be offset upward by the amount of your analyte contained in the matrix. If the *slope* is different, then you have a matrix effect.

The issue of other metabolites is specificity. At some point you have to demonstrate that you can separate the metabolite you are interested in from all other compounds that are likely to be present. Assuming you know what compound you are looking for, you should be able to make a very good guess as to what related compounds are likely to be there. You can run those as standards to establish that they are, in fact, separated. I would then run fairly high-level spikes to confirm that they are still separated in the presence of the matrix.

SPE recoveries are not all that great in general. You might want to consider using an internal standard as well.

It is effect of your sample matrix on slope and intercept of calibration curve compare to calibration curve prepared in pure solvent.

Prepare both and run ANOVA or other stat. tests that can tell you if slopes and intercepts of 2 curves stat. different. If they are, you need to calibrate in std prepared in matrix, or by std additions.

Thanks everyone for all your help!

I have a few extra questions...

Tom: Just to check when you say I should use standards spiked into my matrix do you mean without any cells present (i.e. just 99 ml of milliQ plus 1 ml of fresh media) or should I be adding 1 ml of culture (media plus cells)?

DR: I have been adding an internal standard following elution and prior sample concentration. Once I have my calibration curves sorted I was planning on determining my % recovery by comparing samples I spiked before running through the SPE cartridge with samples spiked following elution. Am I going about this the right way? Or should I be adding an internal standard to the original sample before running through the SPE cartridge?

Thanks again!

Tom: Just to check when you say I should use standards spiked into my matrix do you mean without any cells present (i.e. just 99 ml of milliQ plus 1 ml of fresh media) or should I be adding 1 ml of culture (media plus cells)?

I meant cells included. That said, it wouldn't be a bad idea to do it both ways as long as you're at it. If there *are* matrix effects, that would help you track down the culprit.

DR: I have been adding an internal standard following elution and prior sample concentration. Once I have my calibration curves sorted I was planning on determining my % recovery by comparing samples I spiked before running through the SPE cartridge with samples spiked following elution. Am I going about this the right way? Or should I be adding an internal standard to the original sample before running through the SPE cartridge?

I'd spike the original sample with an internal standard (ideally, an easily separated, close analog of your analyte of interest). That way, you just do all calculations based on peak area (or height) ratios and any small to moderate losses due to extraction inefficiencies cancel out.

Can I emphasise DR's important words: "small to moderate". Internal standards are great, but I worry about the number of methods around the world where no one has noticed that the recovery has gradually slumped and is now about 1%...
The correction in Internal standard calibration is a bit like an extrapolation: the further it has to correct, the less reliable the result, so it's still a good idea to question serious losses. You are right to calculate the recovery of analyte through different steps of extraction.